Kinase control of the tricellular junction protein Gliotactin, and Gliotactin-induced phenotypes in epithelia

This thesis investigates the kinase-mediated regulation of the tricellular junction protein, Gliotactin and signaling pathways involved in Gliotactin overexpression-induced detrimental phenotypes. Tricellular junctions (TCJ) are uniquely placed permeability barriers formed in polarized epithelia where tight junctions in vertebrates or septate junctions in invertebrates from three cells converge. Misregulation of TCJ specific proteins is detrimental to life. However, mechanisms of their localization, maintenance, and potential signaling are largely unknown. Gliotactin is a transmembrane protein unique to TCJ in Drosophila and is essential for the maturation and maintenance of both bicellular and tricellular septate junctions. However, overexpression of Gliotactin leads to the spread of Gliotactin away from the TCJ and disrupts epithelial architecture by signaling for overproliferation, delamination, migration and apoptosis. One mechanism to control Gliotactin is phosphorylation of two highly conserved tyrosine residues and subsequent endocytosis. However, Gliotactin tyrosine phosphorylation also elicits detrimental phenotypes when dysregulated. The kinases involved in Gliotactin phosphorylation had not been broadly investigated prior to this work. We carried out an RNAi screen for phospho-regulators (kinases and some kinase-associated proteins) to determine which could modify the detrimental phenotypes triggered by Gliotactin overexpression. Four suppressors, four partial suppressors, and 53 enhancers were identified by screening 275 RNAi lines covering 164 genes. We determined that Gliotactin overexpression phenotypes involved TNF-JNK, PI3K-Akt signaling pathways and Btk29A. C-terminal Src kinase (Csk), Ret, PI4KIII-α, Skittles and Pkaap were also identified as candidates for further studies. We focused our analysis on Csk and determined Csk is a regulator of Gliotactin endocytosis and plays a role in the regulation of Gliotactin at the TCJ. Although Csk is known as a negative regulator of Src kinases, we identified that the effect of Csk on Gliotactin is independent of Src, and likely occur through an AJ-associated complex. Taken together, this thesis provides novel insights on the function of Csk and identifys other candidate kinases that have the potential to regulate localization and/or signaling events associated with TCJ formation and function.Science, Faculty ofZoology, Department ofGraduat